For bottom-up strategy, proteins are digested into peptides before analyzed by HPLC-MS/MS. However, the large number of peptides not only brings tremendous challenge to chromatography separation, but also might result in false positive identifications. Therefore, intact proteins separation before digestion might be helpful. Recently, Dovichi et al integrated the intact proteins separation by CSE, on-line digestion by pepsin immobilized microreactor, the separation of resulting peptides by MEKC and the protein identified by MS/MS, and showed the promising of the combination of top-down and bottom-up strategies [1]. In our recent work, a novel integrated capillary electrophoresis platform, combining CIEF with monolithic immobilized pH gradient (M-IPG) materials for protein separation, trypsin immobilized microenzymatic reactor for on-line protein digestion, and CZE for peptide separation was established, and successfully applied for protein analysis.
In the integrated platform, a tee unit was used to connect M-IPG column and microreactor, and to supply buffer to improve the compatibility of protein separation and digestion. Another interface was made by Teflon tube to improve the compatibility of microreactor and CZE. By such a platform, a 3-protein-mixture was analyzed, and 15 fractions from M-IPG CIEF were in sequence transferred to microreactor, and further separated by CZE. With collected peptides identified by MALDI-TOF MS, 4-peptide of myoglobin and 7-peptide of BSA were identified by such an integrated M-IPG CIEF-microreactor-CZE platform, which shows the potential of such a platform in high throughput protein analysis.